Since a role of an RNA interference (hereinafter, referred to as ‘RNAi’) had been recognized, it was found that the RNA interference sequence-specifically acts on mRNA in various kinds of mammalian cells (Silence of the transcripts: RNA interference in medicine. J. Mol. Med., 2005, 83: 764-773).
When a long-chain double-stranded RNA is delivered into a cell, the delivered double-stranded RNA is converted into a small interfering RNA (hereinafter, referred to as ‘siRNA’) which is processed to 21 to 23 base pairs (bp) by Dicer endonuclease. siRNA has a short-chain double-stranded RNA having 19 to 27 bases and is coupled to an RNA-induced silencing complex (RISC), whereby a guide (antisense) strand recognizes and degrades a target mRNA to sequence-specifically inhibit expression of a target gene (Nucleic-acid therapeutics: basic principles and recent applications. Nature Reviews Drug Discovery. 2002. 1, 503-514).
The long-chain double-stranded RNA delivered from the outside has a problem of eliciting a non-sequence-specific immune stimulation through interferon expression in a mammal cell; however, it was found that the problem may be overcome by a short-stranded siRNA (Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001. 411, 494-498).
It is known that a chemically synthesized siRNA has a double strand of about 19 to 27 base pairs and consists of a 2-nt(nucleotide) overhang structure at 3′ end, and in order that the double-stranded siRNA expresses an activity, the structure may consist of 3′-hydroxyl groups (OH) and 5′-phosphate groups (PO4) (Effect of asymmetric terminal structures of short RNA duplexes on the RNA interference activity and strand selection. Nucleic Acids Res 1 Oct. 2008: 5812-5821; Strand-specific 5′-O-methylation of siRNA duplexes controls guide strand selection and targeting specificity. RNA 1 Feb. 2008: 263-274).
It is known that a commercialized and synthesized siRNA has a structure in which hydroxyl groups are present at both ends, and when the synthesized siRNA is delivered into a cell, siRNA 5′ end is phosphorylated by a phosphorylation enzyme (kinase) to express functions of siRNA (siRNA function in RNAi: A chemical modification analysis. RNA 2003. 9: 1034-1048).
Bertrand et al. found that as compared to an antisense oligonucleotide (ASO) on the same target gene, siRNA has an effect of significantly inhibiting mRNA expression in vitro and in vivo, and the corresponding effect is maintained for a long time (Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. Biochem. Biophys. Res. Commun. 2002. 296: 1000-1004).
In addition, since siRNA is complementarily coupled to a target mRNA to sequence-specifically regulate an expression of the target gene, a mechanism of the siRNA has an advantage that a target to be capable of being applied may be remarkably increased as compared to the existing antibody-based medical product or chemical material (small molecular drug) (Progress Towards in Vivo Use of siRNAs. MOLECULAR THERAPY. 2006 13(4):664-670).
In order to develop the siRNA as a therapeutic agent despite of excellent effect and variously usable range of the siRNA, the siRNA is required to be effectively delivered into a target cell by improving stability of the siRNA and a cell delivery efficiency (Harnessing in vivo siRNA delivery for drug discovery and therapeutic development. Drug Discov Today. 2006 January; 11(1-2):67-73).
In addition, the siRNA still has a non-specific innate immune stimulation, such that 2-methoxy-, 2-fluoro-substituents have been developed to overcome the non-specific innate immune stimulation.
Since the siRNA is not capable of passing through a hydrophobic phospholipid bilayer of a cell due to negative charges thereof, it is difficult to be delivered into the cell through a simple diffusion.
In order to increase siRNA delivery efficiency in vivo or in vitro, various kinds of cell delivery materials have been developed. Liposomes, cationic surfactants, and the like, are commonly used, and the use of carrier, that is, a fusion method of a gene with liposome or a method of using lipid or a polymer having cations has been known, or a method. of chemically modifying siRNA. or a. method of using conjugate has been known (Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides. Nucleic Acids Res. 2008 July; 36(12):4158-71).
Since the siRNA is not capable of passing through a hydrophobic phospholipid bilayer of a cell due to negative charges thereof, it is difficult to be delivered into the cell through a simple diffusion, such that in order to overcome the difficulty, methylphosphonate or peptide nucleic acid (PNA) is used in a basic binding structure of the siRNA. In addition, a carrier is used, for example, a fusion method of a gene with liposome or a method of using lipid. or a polymer having cations is used (Chemically modified siRNA: tools and applications. Drug Discov. Today. 2008 October; 13(19-20):842-855).
Among them, as a method of using a nanocarrier, a method of using various polymers such as liposome, cationic polymer complex, and the like, is to carry siRNA on a nanocarrier by formation of nanoparticles to deliver siRNA. Among the methods of using nanocarriers, a method of using polymeric nanoparticle, polymer micelle, lipoplex, or the like, is mainly used, wherein the lipoplex consists of cationic lipid to interact with anionic lipid of endosome of a cell, thereby eliciting a destabilization effect of the endosome to deliver the siRNA into a cell (Mechanism of oligonucleotide release from cationic liposomes. Proc. Natl. Acad. Sci. USA. 1996 Oct. 15; 93(21):11493-8).
In addition, it is known that chemical materials, and the like, are connected to end portions of a siRNA passenger (sense strand) to provide increased pharmacokinetics characteristics and high efficacy may be induced in vivo (Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature. 2004 Nov. 11; 432(7014):173-8). Here, stability of the siRNA may vary depending on properties of the chemical materials bound to ends of the siRNA sense (passenger) or antisense (guide) strand. For example, a siRNA having a polymer compound such as polyethylene glycol (PEG) conjugated thereto interacts with an anionic phosphate group of siRNA in the presence of cationic materials to form a complex, thereby being a siRNA carrier having an improved stability (Local and systemic delivery of VEGF siRNA using polyelectrolyte complex micelles for effective treatment of cancer. J Control Release. 2008 Jul. 14; 129(2):107-16). In particular, micelle consisting of polymer complexes has an extremely small size, significantly uniform distribution, and is spontaneously form, thereby being easy to manage quality of formulation and secure reproducibility, as compared to other systems used as a drug delivery vehicle, such as microsphere, nanoparticle, and the like.
Recently, in order to improve an intracellular delivery efficiency of siRNA, technology of using a siRNA conjugate in which hydrophilic material which is a biocompatible polymer (for example, polyethylene glycol (PEG)) is conjugated to the siRNA by a simple covalent bond or a linker-mediated covalent bond, to thereby secure stability of siRNA and have effective cell membrane permeability was developed (see Korean Patent Publication No. 883471).
However, the chemical modification of siRNA and the conjugation with the polyethylene glycol (PEG) (PEGylation) still has disadvantages that stability in vivo is low and delivery into a target organ is not smooth. In chemical modification of siRNA, a bond to RISC without modification at 5′ end of an antisense (guide) strand recognizing a target mRNA is significantly important to initiation of RNAi mechanism. In a case of a sense (passenger) strand, through the existing research, functions of siRNA are confirmed even in a case where the conjugates are bound to both of ends, such that the sense (passenger) strand is utilized for a conjugate bond (siRNA Conjugate Delivery Systems. Bioconjugate Chem., 2009, 20 (1), pp 5-14).
In a case of a double-stranded oligo RNA structure in which the hydrophilic materials and the hydrophobic materials are bound to the double-stranded oligo RNA, self-assembling nanoparticles are formed by a hydrophobic interaction of the hydrophobic materials, wherein the self-assembling nanoparticle is referred to as ‘SAMiRNA’ (Korean Patent Laid-Open Publication No. 2009-0042297).
The technology of forming the self-assembling nanoparticles (SAMiRNA) by binding the hydrophobic materials and the hydrophilic materials to an end of the double-stranded oligo RNA has a possibility of RNA strand bias of a double-stranded oligo RNA, that is, RNAi functions may be inhibited depending on a position where the hydrophilic materials and the hydrophobic materials are bound to the end. Therefore, a technology of delivering a double-stranded oligo RNA capable of effectively permeating a cell membrane without inhibiting the functions of the double-stranded oligo RNA through optimization of the double-stranded oligo RNA structure is inevitably required to be developed.